The P1 protein of wheat yellow mosaic virus exerts RNA silencing suppression activity to facilitate virus infection in wheat plants

Abstract

SUMMARYWheat yellow mosaic virus (WYMV) causes severe wheat viral disease in Asia. However, the viral suppressor of RNA silencing (VSR) encoded by WYMV has not been identified. Here, the P1 protein encoded by WYMV RNA2 was shown to suppress RNA silencing in Nicotiana benthamiana. Mutagenesis assays revealed that the alanine substitution mutant G175A of P1 abolished VSR activity and mutant Y10A VSR activity remained only in younger leaves. P1, but not G175A, interacted with gene silencing‐related protein, N. benthamiana calmodulin‐like protein (NbCaM), and calmodulin‐binding transcription activator 3 (NbCAMTA3), and Y10A interacted with NbCAMTA3 only. Competitive Bimolecular fluorescence complementation and co‐immunoprecipitation assays showed that the ability of P1 disturbing the interaction between NbCaM and NbCAMTA3 was stronger than Y10A, Y10A was stronger than G175A. In vitro transcript inoculation of infectious WYMV clones further demonstrated that VSR‐defective mutants G175A and Y10A reduced WYMV infection in wheat (Triticum aestivum L.), G175A had a more significant effect on virus accumulation in upper leaves of wheat than Y10A. Moreover, RNA silencing, temperature, and autophagy have significant effects on the accumulation of P1 in N. benthamiana. Taken together, WYMV P1 acts as VSR by interfering with calmodulin‐associated antiviral RNAi defense to facilitate virus infection in wheat, which has provided clear insights into the function of P1 in the process of WYMV infection.

The P2 protein of wheat yellow mosaic virus acts as a viral suppressor of RNA silencing to facilitate virus infection in wheat plants

“…It was recently found that RNA silencing can be regulated by the signal cascade of calmodulin (CaM) protein binding with calmodulin-binding transcription activator 3 (CAMTA3) protein; the CaM– CAMTAs interaction upregulates transcription of RNA-silencing related genes such as those encoding dicer1 (DCL1), bifunctional nuclease 2 (BN2), Argonaute protein (AGO), and RNA-dependent RNA polymerase 6 (RDR6), which enhance the resistance of Nicotiana benthamiana plants (Wang et al, 2021; Wang et al, 2022). Our laboratory research showed that WYMV P1 protein also has viral suppressor of RNA silencing (VSR) activity, and plays a suppressor role by interfering with calmodulin-related antiviral RNAi defense, confirming that its VSR activity contributes to the systemic infection of WYMV in wheat (Chen et al, 2023). The P2 proteins of BaMMV, OMV, and BaYMV may be related to fungal transmission (Jacobi et al, 1995; Peerenboom et al, 1992; Zheng et al, 2002).…”

“…The qRT-PCR results showed that the gene for CaM was the most significantly downregulated; it was recently reported that VSRs exert suppressor activity by directly interacting with CaM, thereby interfering with the CaM–CAMTA3 interaction (Chen et al, 2023; Wang et al, 2021; Wang et al, 2022). Therefore, Y2H and BiFC were used to verify the P2–CaM interaction (Figure 3; Supporting Figure 7).…”

“…Agrobacterium co-infiltration experiments showed that P2 had relatively strong local VSR activity in N. benthamiana leaves at the four-leaf stage, but weak at the eight-leaf stage (Figure 1B and C). The WYMV P2 was similar to P1 in that it had similar local VSR activity and only exhibited strong suppressor activity in the younger N. benthamiana leaves, which further explains why wheat seedlings need to be inoculated with WYMV at the one to two leaf stage (Chen et al, 2023; Zhang et al, 2021; Shang et al, 2002). One significant difference between them was that the P2 truncated mutants had local VSR activity (Figure 2A–C), while the P1 truncated mutants tested lost local VSR activity (Chen et al, 2023).…”

“…The WYMV P2 was similar to P1 in that it had similar local VSR activity and only exhibited strong suppressor activity in the younger N. benthamiana leaves, which further explains why wheat seedlings need to be inoculated with WYMV at the one to two leaf stage (Chen et al, 2023; Zhang et al, 2021; Shang et al, 2002). One significant difference between them was that the P2 truncated mutants had local VSR activity (Figure 2A–C), while the P1 truncated mutants tested lost local VSR activity (Chen et al, 2023). Amino acid sequence alignment of Bymovirus P2 revealed 13 conserved amino acid residues (Figure 2D), and VSR activity screening of the 13 point mutants showed that conserved proline 70 and glycine 230 in P2 were essential for RNA-silencing suppressor activity (Figure 2E and F).…”

“…Viruses have developed a variety of mechanisms to overcome antiviral RNA silencing. In general, the mechanisms of viral silencing suppressors can be divided into two broad categories in the downstream of the RNA-silencing pathway (Brodersen and Voinnet, 2006; Chen et al, 2023; Li et al, 2019; Marco et al, 2013). The first kind of VSRs act on proteins associated with RNA silencing, such as the P38 protein of turnip crinkle virus (TCV) (Schott et al, 2012; Thomas et al, 2003), the P1 protein of sweet potato mild mottle virus (Giner et al, 2010) interacts with AGO1 to inhibit RNA silencing, V2 encoded by tomato yellow leaf curl virus interacts with SGS3 and exerts suppressor activity (Glick et al, 2008), and P6 protein encoded by rice yellow stunt virus interacts with RDR6 to inhibit transgene-induced systemic silencing (Hongyan et al, 2013).…”